The norepinephrine (NE) transporter (NET) mediates the removal of NE from synaptic spaces and is a major target for antidepressants, amphetamine and cocaine. Previously, we have shown that syntaxin 1A (SYN 1A) supports human NET (hNET) cell surface expression, that hNET/SYN 1A interactions are direct and mediated by the hNET N-terminus, and that the hNET/SYN 1A association limits substrate-induced hNET-associated currents [Sung, U., Apparsundaram, S., Galli, A., Kahlig, K.M., Savchenko, V., Schroeter, S., Quick, M.W., Blakely, R.D., 2003. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J. Neurosci. 23, 1697-1709]. These data raise the possibility that the hNET N-terminus, and potentially its interaction with SYN 1A, might regulate other hNET conductance states, including the hNET-mediated leak current. Importantly for monoamine transporters, the leak conductance has been shown to play a critical role in regulating cell membrane potential and possibly neuronal excitability [Quick, M.W., 2003. Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537-549]. Here we demonstrate that deletion of the binding domain for SYN 1A in the NET N-terminus robustly enhances the NET-mediated leak current as well as its selectivity for Cl- permeation under particular intracellular ionic compositions. In addition, we show that the NET N-terminus coordinates the ability of intracellular Na + and Cl- to regulate the leak conductance. These data suggest that the NET N-terminus regulates and defines the ionic specificity of the NET-mediated leak current. © 2005 Elsevier Ltd. All rights reserved.